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Article in press - uncorrected proof Clin Chem Lab Med 2006;44(9):1146–1155 2006 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2006.212

2006/251

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C

International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)1), 2) Scientific Division, Committee on Reference Systems for Enzymes (C-RSE)3) Part 8. Reference procedure for the measurement of catalytic concentration of a-amylase wa-Amylase: 1,4-a-D-glucan 4-glucanohydrolase (AMY), EC 3.2.1.1x

Gerhard Schumann1,*, Ryoji Aoki2, Carlo A. Ferrero3, Glenn Ehlers4, Georges Fe´rard5, F.-Javier Gella6, Poul Jørgen Jørgensen7, Takahashi Kanno8, Art Kessner9, Rainer Klauke1, Hans-Joachim Kytzia10, Jean-Marc Lessinger5, W. Gregory Miller11, Rolf Nagel10, Jean Pauwels12, Heinz Schimmel12, Lothar Siekmann13, Gerhard Weidemann14, Kiyoshi Yoshida2 and Ferruccio Ceriotti3 The exclusive for all languages and countries is vested in the International Federation of Clinical Chemistry and Laboratory Medicine. 2) IFCC Sections printed in J. Clin. Chem. Clin. Biochem. are listed in the Cumulative Index, which appeared in connection with the contents of this journal in Volume 27, 1989 and since 1991 have been printed in (Eur.) J. Clin. Chem. Clin. Biochem. IFCC 1991/1 Vol. 29, 435–457 IFCC 1991/2 Vol. 29, 531–535 IFCC 1991/3 Vol. 29, 577–586 IFCC 1991/4 Vol. 29, 767–772 IFCC 1992/1 Vol. 30, 901–905 IFCC 1994/1 Vol. 32, 639–655 IFCC 1995/1 Vol. 33, 247–253 IFCC 1995/2 Vol. 33, 399–404 IFCC 1995/3 Vol. 33, 623–625 IFCC 1995/4 Vol. 33, 627–636 IFCC 1995/5 Vol. 33, 637–660 IFCC 1997/1 Vol. 35, 317–344 IFCC 1997/2 Vol. 35, 345–349 IFCC 1997/3 Vol. 35, 805–831 IFCC 1997/4 Vol. 35, 833–843 For IFCC sections printed in Clin. Chem. Lab. Med. since 1998, please visit the link http://degruyter.com/journals/ extenza, where they are freely accessible. 3) Members: L. Siekmanna (DE), T. Kannoa (JP), F. Ceriottib (IT), G. Fe´rardb (FR), F. Canaliasc (ES), P.J. Jørgensenc (DK), W.G. Millerc (USA), D. Kangd (JP), G. Schumann (DE); amember until 2004, bmember until 2005, cmember since 2005, d member since 2006. *Corresponding author: Prof. Gerhard Schumann, Klinische Chemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30623 Hannover, Germany Phone: q49-511-5322523, Fax: q49-511-5325671, E-mail: [email protected] Received for publication June 29, 2006 1)

1

Klinische Chemie, Medizinische Hochschule Hannover, Hannover, Germany 2 Research & Development, Asahi Chemical Industry Co., Tokyo, Japan 3 Laboratorio Standardizzazione, Diagnostica e Ricerca San Raffaele s.p.a., Milano, Italy 4 Ortho-Clinical Diagnostics, Rochester, NY, USA 5 Laboratoire de Biochimie applique´e, Faculte´ de Pharmacie, Universite´ Louis Pasteur, Illkirch, France 6 BioSystems, S.A., Barcelona, Spain 7 Department of Clinical Chemistry, Odense University Hospital, Odense, Denmark 8 Department of Laboratory Medicine, Hamamatsu University, School of Medicine, Japan 9 Beckman Coulter, Inc., Brea, CA, USA 10 Roche Diagnostics GmbH, Mannheim, Germany 11 Virginia Commonwealth University, Richmond, VA, USA 12 European Commission, Joint Research Centre, Institute for Reference Materials and Measurements, Geel, Belgium 13 Institut fu¨r Klinische Biochemie, Universita¨t Bonn, Bonn, Germany 14 Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Klinikum der Stadt, Nu¨rnberg, Germany

Abstract This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of g-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of g-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 378C. The procedure described here is deduced from the previously described 308C IFCC reference method. Differences are tabulated and commented on. Clin Chem Lab Med 2006;44:1146–55.

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Keywords: a-amylase; IFCC reference procedure; preliminary upper and lower reference limits. Abbreviations: AMY, a-amylase; EPS, 4,6-ethylidene(G1)-4-nitrophenyl(G7)-a-(1™4)-D-maltoheptaoside; G, a-(1™4)-D-glucopyranosyl-; 4-NP, 4-nitrophenoxide; G7-4-NP, 4-nitrophenyl-a-(1™4)-D-maltoheptaoside; IRMM, Institute for Reference Materials and Measurements.

Temperature Wave length Band width Light path Incubation time Delay time Measurement interval Readings (measurement points)

37.08C"0.18C 405 nm "1 nm F2 nm 10.00 mm "0.01 mm 60 s 180 s 180 s G6

Reagents

Introduction This paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C and the certification of reference prepartions (1–7).

Reaction principle

a-Amylase

EPSqH2O

Table 2 Conditions for the measurement of a-amylase.

™

4,6-Ethylidene-G x q4-Nitrophenyl-G (7–x)

4-Nitrophenyl-G (7–x)

1. N-2-Hydroxyethylpiperazine-N9-ethanesulfonic acid wHEPESx (C8H18N2O4S), Mrs238.31 2. 4,6-Ethylidene(G1)-4-nitrophenyl(G7)-a-(1™4)-Dmaltoheptaoside wEPSx, (C50H77NO38), Mrs1300.1 3. a-Glucosidase (EC 3.2.1.20) 4. Sodium chloride (NaCl), Mrs58.44 5. Calcium chloride dihydrate (CaCl2Ø2H2O), Mrs147.02 6. Sodium hydroxide solution (NaOH), Mrs40.00, 0.2 molØl –1 7. Bovine serum albumin, fraction V, Mrs68000 8. Aqueous sodium chloride solution (NaCl), Mrs58.44, 0.154 molØl –1 9. Water (analytical grade), Mrs18.02

a-Glucosidase

q(7–x) H2O

™

(7–x) Glucose q4-Nitrophenoxide

Specimens Calibration materials, control specimens and human sera.

Measurement conditions Concentrations in the final reaction mixture and the measurement conditions are listed in Tables 1 and 2. Note: Compliance with the prescribed tolerances for the parameters for temperature, pH, light path and wavelength is confirmed if the combined expanded uncertainty of the calibration is equal to or less than the tolerances and if the range of uncertainty of the calibration overlaps the prescribed tolerance intervals.

Reagents of the highest purity must be used. The quality of the reagents has to be proved by certificates of analysis from the suppliers. If a chemical is suspected of containing impurities affecting the catalytic activity of the analyte, further investigations must be performed, e.g., comparisons with products from different manufacturers and different lots. It is recommended to use reagents that have been tested and approved in comparisons. EPS: Contamination of the substrate EPS by 4-nitrophenyl-a-D-maltoheptaoside (G7-4-NP) may cause a prolonged lag phase and may then interfere with the determination of the catalytic concentration of a-amylase. The mass fraction of G7-4-NP in EPS is limited to 0.001. If the mass fraction of G7-4-NP is unknown to the user or not clearly specified in a certificate, the mass concentration of G7-4-NP has to be determined (see Appendix 2). a-Glucosidase: Only enzyme preparations of a-glucosidase that split all glucosidic bonds of the a-amy-

Table 1 Concentrations in the final complete reaction mixture for the measurement of a-amylase. N-2-Hydroxyethylpiperazine-N9-ethanesulfonic acid pH (378C) 4,6-Ethylidene(G1)-4-nitrophenyl(G7)-a-(1™4)-D-maltoheptaoside Sodium chloride Calcium chloride a-Glucosidase (378C) Volume fraction of sample

50 mmolØl –1 7.00"0.03 5 mmolØl –1 70 mmolØl –1 1 mmolØl –1 135 mkatØl –1 (8100 UØl –1)* 0.0323 (1:31)

*Uninhibited catalytic concentration. Note: The indicated catalytic concentration of a-glucosidase shall be measured in the final complete reaction mixture if 9 gØl –1 (154 mmolØl –1) sodium chloride solution is used as the sample (no inhibition by the sample matrix). Note: Besides the substances listed in Table 1, the final complete reaction mixture contains 0.1 gØl –1 albumin, which is originally a component of Solution 3. The presence of albumin in the Reaction Solution and consequently in the final complete reaction mixture stabilizes the a-glucosidase.

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Schumann et al.: IFCC primary reference procedures

lase reaction products are suitable (e.g., from Bacillus stearothermophilus). Bovine serum albumin: The bovine serum albumin used must be free from proteases as declared in the manufacturer’s specifications.

A deviation in pH of 0.01 causes a change in the kinetics of approximately 1%. Therefore, the pH has to be adjusted very accurately.

Charts for the adjustment and control of pH Calibration of the pH meter The molar absorption coefficient of the indicator strongly depends on the pH. Therefore the calibration of the pH-meter and the adjustments of the pH of solution 2 and the starting reagent solution must be very accurate. At least two standard buffer solutions have to be used for the calibration procedure. The standard buffer solutions must have certified values in the range from pH 6 to pH 8 and shall include the adjusting pH of the reagent solutions. The uncertainty of the certified pH has to be F0.01 pH. Note: The use of standard buffer solutions containing potassium dihydrogen phosphate (anhydrous) and disodium hydrogen phosphate (anhydrous) is recommended. Appropriate certified standard reference buffer solutions traceable to national or international standard reference materials are commercially available. Note: The molar absorption coefficient of the indicator (4-nitrophenoxide) strongly depends on the pH.

Procedure for the adjustment of pH at temperatures diverging from 378C Both the thermometer and the pH electrode are suspended in the mixed solution simultaneously. The stirred solution is then titrated to the pH listed in Tables 3 and 4 for the actual temperature measured. The speed of agitation should be the same during calibration and control of the pH meter and adjustment of the pH of the reagent solutions. The pH electrode should be positioned in the center of the stirred solution. The fact that the temperature can change during the titration must be taken into account. For this reason, the temperature in the proximity of the target value should be controlled again and the target pH has to be corrected if necessary. The same applies for the adjustment of the temperature compensation of the pH meter.

Table 3 Dependence of the pH of Solution 2 on temperature. Temperature, 8C

pH

Temperature, 8C

pH

Temperature, 8C

pH

15.00 15.25 15.50 15.75 16.00 16.25 16.50 16.75 17.00 17.25 17.50 17.75 18.00 18.25 18.50 18.75 19.00 19.25 19.50 19.75 20.00 20.25 20.50 20.75 21.00 21.25 21.50 21.75 22.00 22.25 22.50 22.75 23.00 23.25

7.254 7.251 7.248 7.245 7.242 7.239 7.236 7.233 7.230 7.227 7.224 7.221 7.218 7.215 7.212 7.209 7.206 7.203 7.200 7.197 7.194 7.192 7.189 7.186 7.183 7.180 7.177 7.174 7.171 7.168 7.165 7.162 7.159 7.157

23.50 23.75 24.00 24.25 24.50 24.75 25.00 25.25 25.50 25.75 26.00 26.25 26.50 26.75 27.00 27.25 27.50 27.75 28.00 28.25 28.50 28.75 29.00 29.25 29.50 29.75 30.00 30.25 30.50 30.75 31.00 31.25 31.50 31.75

7.154 7.151 7.148 7.145 7.142 7.139 7.136 7.133 7.130 7.128 7.125 7.122 7.119 7.116 7.113 7.110 7.107 7.105 7.102 7.099 7.096 7.093 7.090 7.087 7.085 7.082 7.079 7.076 7.073 7.070 7.068 7.065 7.062 7.059

32.00 32.25 32.50 32.75 33.00 33.25 33.50 33.75 34.00 34.25 34.50 34.75 35.00 35.25 35.50 35.75 36.00 36.25 36.50 36.75 37.00 37.25 37.50 37.75 38.00 38.25 38.50 38.75 39.00 39.25 39.50 39.75 40.00

7.056 7.053 7.051 7.048 7.045 7.042 7.039 7.036 7.034 7.031 7.028 7.025 7.022 7.020 7.017 7.014 7.011 7.008 7.006 7.003 7.000 6.997 6.994 6.992 6.989 6.986 6.983 6.981 6.978 6.975 6.972 6.969 6.967


Table 4 Dependence of the pH of the starting reagent solution on temperature. Temperature (8C)

pH

Temperature (8C)

pH

Temperature (8C)

pH

15.00 15.25 15.50 15.75 16.00 16.25 16.50 16.75 17.00 17.25 17.50 17.75 18.00 18.25 18.50 18.75 19.00 19.25 19.50 19.75 20.00 20.25 20.50 20.75 21.00 21.25 21.50 21.75 22.00 22.25 22.50 22.75 23.00 23.25

7.270 7.267 7.264 7.261 7.258 7.255 7.252 7.249 7.246 7.243 7.240 7.237 7.234 7.231 7.227 7.224 7.221 7.218 7.215 7.212 7.209 7.206 7.203 7.199 7.196 7.193 7.190 7.187 7.184 7.181 7.177 7.174 7.171 7.168

23.50 23.75 24.00 24.25 24.50 24.75 25.00 25.25 25.50 25.75 26.00 26.25 26.50 26.75 27.00 27.25 27.50 27.75 28.00 28.25 28.50 28.75 29.00 29.25 29.50 29.75 30.00 30.25 30.50 30.75 31.00 31.25 31.50 31.75

7.165 7.162 7.158 7.155 7.152 7.149 7.146 7.143 7.139 7.136 7.133 7.130 7.127 7.124 7.120 7.117 7.114 7.111 7.108 7.105 7.102 7.099 7.095 7.092 7.089 7.086 7.083 7.080 7.077 7.074 7.071 7.068 7.064 7.061

32.00 32.25 32.50 32.75 33.00 33.25 33.50 33.75 34.00 34.25 34.50 34.75 35.00 35.25 35.50 35.75 36.00 36.25 36.50 36.75 37.00 37.25 37.50 37.75 38.00 38.25 38.50 38.75 39.00 39.25 39.50 39.75 40.00

7.058 7.055 7.052 7.049 7.046 7.043 7.040 7.037 7.034 7.032 7.029 7.026 7.023 7.020 7.017 7.014 7.011 7.008 7.006 7.003 7.000 6.997 6.994 6.992 6.989 6.986 6.984 6.981 6.978 6.976 6.973 6.970 6.968

Preparation of solutions The mass given for compounds for the preparation of solutions refers to 100% content. If the content of the reagent chemical employed is less (e.g., yz %), the amount equivalent to the given mass is calculated using a factor: Fcontents

100 yz

Impurities in the water for the preparation of the reagent solutions for a-amylase measurements may cause inhibition of the catalytic activity. Guidelines describing the preparation and testing of reagent Table 5 Analytical system for the measurement of a-amylase. 2.000 ml Reaction solution Equilibrate to 37.08C 0.080 ml Sample Mix thoroughly and incubate for 60 s. At the end of the incubation time, the temperature of the solution in the cuvette shall have reached 37.08C 0.400 ml Starting reagent solution Mix thoroughly, wait 180 s and monitor time and absorbance for an additional 180 s

water are published elsewhere (9). However, standards prescribing the quality of water specifically for enzyme measurements are not yet defined. In the case of suspected influence of the water on determination of the catalytic activity concentration, systematic investigations have to be performed. The expanded (ks2) combined uncertainty (normally distributed) of each weighing procedure (including the uncertainty of the purity of the substance) shall be F1.5% and the mass on the display of the balance shall not differ from the target mass by more than "0.5%. Solution 1 6.14 g (417.5 mmolØl –1) calcium chloride, dihydrate. • • • •

Dissolve in approximately 80 ml of water. Transfer to a 100-ml volumetric flask. Equilibrate the volumetric flask and water to 208C. Fill with water (208C) up to the calibration mark of the volumetric flask. Stability at 208C: 3 months.

Solution 2 3.10 g (52.10 mmolØl –1) N-2-hydroxyethylpiperazineN9-ethanesulfonic acid.



1.26 g (86.13 mmolØl –1) sodium chloride. • Dissolve in approximately 200 ml of water. • Add 0.75 ml of solution 1. • Adjust to pH (378C) 7.00 with 0.2 molØl –1 sodium hydroxide solution. • Transfer to a 250-ml volumetric flask. • Equilibrate the volumetric flask and water to 208C. • Fill with water (208C) up to the calibration mark of the volumetric flask. Stability at 28C–88C: 5 weeks.

shortly before the measurement procedure. The remaining volume of the starting reagent solution should be stored at 28C–88C. Pipette the volumes listed in Table 5 one after another into the cuvette. A high-performance spectrometer and high-accurate volumetric devices shall be used. The inaccuracy of the kinetic photometric measurements and the inaccuracy of the volume fraction of sample shall be determined by test procedures with known standard uncertainties.

Diluent for reagent enzymes

Reagent blank rate

1.20 g bovine albumin. 0.900 g (154 mmol l –1) NaCl.

To determine the reagent blank rate, the sample is replaced by 9 gØl –1 (154 mmolØl –1) sodium chloride solution. The measurement procedure is then carried out as described above. The initial absorbance should not exceed 0.35 and the change in absorbance of the reagent blank rate should be less than 3.3=10 –5 s –1 (0.002 min –1). Otherwise, sources of contamination by salivary amylase should be identified and excluded or the purity of the reagents must be reassessed.

• • • •

Dissolve in approximately 80 ml of water. Transfer to a 100-ml volumetric flask. Equilibrate the volumetric flask and water to 208C. Fill with water (208C) up to the calibration mark of the volumetric flask. Stability at 28C–88C: at least 3 months.

Solution 3

Sample blank rate

16.9 mkatØl –1 (1014 kUØl –1) a-glucosidase at 378C. • Determine the catalytic a-glucosidase concentration according to Appendix 1. • Reconstitute the lyophilized a-glucosidase with a volume of the Diluent for Reagent Enzymes to obtain a catalytic concentration of the reconstituted material of 16.9 mkatØl –1 (1014 kUØl –1) at 378C. • Freeze the enzyme solution in portions of 0.25 ml at –258C. Stability at y258C: at least 6 months. Reaction solution • Mix 25 ml of solution 2 with 0.25 ml of solution 3. Stability at 28C–88C: 2 weeks. Starting reagent solution 1.01 g (31.00 mmolØl –1) 4,6-ethylidene(G1)-4-nitrophenyl(G7)-a-(1™4)-D-maltoheptaoside. 0.310 g (52.10 mmolØl –1) N-2-hydroxyethylpiperazineN9-ethanesulfonic acid. • Dissolve in approximately 20 ml of water. • Adjust to pH (378C) 7.00 with 0.2 molØl –1 sodium hydroxide solution. • Transfer to a 25-ml volumetric flask. • Equilibrate the volumetric flask and water to 208C. • Fill with water (208C) up to the calibration mark of the volumetric flask. Stability at 28C–88C: 2 weeks.

Measurement procedure Equilibrate only an adequate volume (f0.6 ml per measurement) of the starting reagent solution at 378C

For determination of the sample blank rate, the starting reagent solution is replaced by 9 gØl –1 (154 mmolØl –1) sodium chloride solution. The measurement procedure is then carried out as described above. Note: The sample blank is determined and documented, but not taken into account for calculation of the catalytic concentration of a-amylase in control sera and calibrators. If the value of the sample blank exceeds 1% of total a-amylase, a warning should be given that the material is not appropriate for calibration. Note: The reagent blank for the sample blank is determined by replacing the starting reagent solution and the sample by 9 gØl –1 sodium chloride solution. Note: Omission of the starting reagent means omission of the substance that forms the indicator. Therefore, interference by the sample matrix on the indicator reaction is not identified. Upper limit of the measurement range If the change in absorbance exceeds 0.0039 s –1 (0.235 min –1) in the measurement interval, an analytical portion of the sample must be diluted with 9 gØl –1 (154 mmolØl –1) aqueous sodium chloride solution and the measurement procedure must be repeated with the diluted sample. The value obtained must then be multiplied by the corresponding dilution factor. Sources of error The catalytic concentration of a-glucosidase in the final complete reaction mixture is differently inhibited by the specific matrix of the specimen investigated (see the respective comments in Appendix 3). Therefore, determination of the matrix-dependent inhibition


of a-glucosidase is necessary for each material investigated (see Appendix 1). Calcium complexing agents reduce the available concentration of this essential component and can lead to reduced measurement values. Contamination by salivary amylase (e.g., by finger contact with the solutions or the inner sides of the solution containers) leads to falsely increased catalytic concentrations of a-amylase or to increased reagent blank rates.

Calculation The temporal change in absorbance (s –1) is calculated using regression analysis (least squares method). After subtraction of the reagent blank rate (s –1), the corrected change in absorbance (s –1) is multiplied by the factor Fs3063 wmeasurement at 405 nm, ´405(4-NP)s1012 m2Ømol –1x Note: Use of the molar absorption coefficient ´405(4NP)s1012 m2Ømol –1 is recommended by IFCC and IRMM (10). The catalytic concentration of a-amylase is calculated in mkatØl –1. Change in absorbance (s –1) after correction DA Amylase for the reagent blank rate (s –1) Dt bAmylase

Catalytic concentration of a-amylase (mkatØl –1)

bAmylases 3063

DA Amylase Dt

The catalytic concentration in mkatØl –1 can be converted to UØl –1 by multiplication by the factor fs60.

Preliminary upper and lower reference limits

0.52 mkatØl –1 (0.42 mkatØl –1 –0.60 mkatØl –1) 31 UØl –1 (25 UØl –1 –36 UØl –1) Upper reference limit* (and 90% confidence interval for the 97.5th percentile) 1.78 mkatØly1 (1.71 mkatØl –1 –2.00 mkatØl –1) 107 UØly1 (103 UØl –1 –120 UØl –1) *The lower and upper reference limits are the 2.5th and 97.5th percentiles of the reference cohorts. Values in parentheses are the 90% confidence intervals for the 2.5th and 97.5th percentiles.

Appendix 1: Determination of the catalytic concentration of a-glucosidase under the reaction conditions of the procedure for a-amylase

Additional reagents 4-Nitrophenyl-a-D-glucopyranoside (C12H15NO8) Mrs301.26

Solution A 1.24 g (52.08 mmolØl –1) N-2-hydroxyethylpiperazineN9-ethanesulfonic acid. 0.419 g (71.63 mmolØl –1) NaCl. • Dissolve in approximately 80 ml of water. • Add 0.25 ml of solution 1 for the procedure for aamylase (calcium chloride stock solution). • Adjust pH (378C) to 7.00 with 0.2 molØl –1 sodium hydroxide solution. • Transfer to a 100-ml volumetric flask. • Equilibrate the volumetric flask and water to 208C. • Fill with water (208C) to the calibration mark of the volumetric flask. Stability at 28C–88C: 5 weeks.

Reference values Preliminary reference intervals were obtained in two different reference laboratories by measuring a-amylase activity in serum samples from two reference cohorts (146 males and 89 females). The reference cohorts comprised serum samples from employees of an annual wellbeing check (first cohort) and from blood donors (second cohort). Samples were excluded from the reference cohort if the concentration at least one of the following quantities in serum exceeded the corresponding upper reference limit: creatinine, glucose, AST, ALT, GGT (and/or ALP) and lipase (cohort 2 only). No significant differences (t-test, p-0.05) could be observed between the two reference cohorts or between samples from males and females. The preliminary reference limits for men and women (G17 years) are: Lower reference limit* (and 90% confidence interval for the 2.5th percentile)

Solution B 0.900 g (154.0 mmolØl –1) sodium chloride. • • • •

Dissolve in approximately 80 ml of water. Transfer to a 100-ml volumetric flask. Equilibrate the volumetric flask and water to 208C. Fill with water (208C) to the calibration mark of the volumetric flask. Stability at 208C: 3 months.

Reaction solution for a-glucosidase 0.0392 g (5.208 mmolØl –1) 4-nitrophenyl-a-D-glucopyranoside • Transfer to a 25-ml volumetric flask and dissolve in approximately 20 ml of solution A. • Equilibrate the volumetric flask and solution A to 208C.



Table 6 Concentrations in the final complete reaction mixture for the measurement of a-glucosidase. N-2-HydroxyethylpiperazineN9-ethanesulfonic acid pH (378C) 4-Nitrophenyl-a-D-glucopyranoside Sodium chloride Calcium chloride Volume fraction of sample

50 mmolØl –1 7.00"0.03 5 mmolØl –1 70 molØl –1 1 mmolØl –1 0.0320 (1:31.25)

Table 7 Conditions for the measurement of a-glucosidase. Temperature Wave length Band width Light path Incubation time Delay time Measurement interval Readings (measurement points)

Table 8 Analytical a-glucosidase.

system for

37.08C"0.18C 405 nm"1 m F2 nm 10.00 mm"0.01 mm 60 s 180 s 180 s G6

the measurement of

2.400 ml Reaction solution for a-glucosidase Equilibrate to 378C 0.080 ml Solution B Mix thoroughly and wait 60 s 0.020 ml Solution of a-glucosidase diluted with the Diluent for Reagent Enzymes Mix thoroughly, wait 180 s and monitor time and absorbance for an additional 180 s Note: Solution B here replaces the sample in the a-amylase measurement system.

• Fill with solution A (208C) up to the calibration mark of the volumetric flask. Stability at 28C–88C: 5 days.

Measurement conditions Concentrations in the reaction mixture and measurement conditions are listed in Tables 6 and 7.

Preparation of a-glucosidase 1. Weight approximately 10 mg of the lyophilized enzyme (record the weighed mass; m) and reconstitute with 1000 ml of Diluent for Reagent Enzymes for the procedure for a-amylase. 2. Dilute 50 ml of the reconstituted a-glucosidase with 1000 ml of the Diluent for Reagent Enzymes. The dilution of the first step is 1:21. 3. Dilute 50 ml of the solution from step 1 with 1000 ml of the Diluent for Reagent Enzymes. The dilution of the second step is 1:21. The total dilution after step 1 and step 2 is 1:441. The dilution factor is thus 441. Note: The indicated dilutions in step 1 and step 2 are appropriate for lyophilized a-glucosidase containing

0.40–1.65 mkatØmg –1 (25–100 UØmg –1). The preparation of lyophilized a-glucosidase may necessitate other dilutions. The dilution factor must be changed corresponding to the dilution.

Measurement procedure The catalytic concentration of a-glucosidase is determined under reaction conditions very close to those of the procedure for a-amylase. The reaction is started with the diluted solution of a-glucosidase. Pipette the volumes indicated in Table 8 sequentially into the cuvette.

Reagent blank rate To determine the reagent blank rate, the diluted solution of a-glucosidase is replaced by the Diluent for Reagent Enzymes. The measurement procedure is then carried out as described above.

Calculation The temporal change in absorbance (s –1) is calculated using regression analysis (least squares method). The reagent blank rate is subtracted. The corrected change of absorbance is used for the calculation of the catalytic mass concentration: Calculation of the catalytic mass concentration of a-glucosidase lyophilizate powder: Fdilution12350 blyos

DA Dt

1000 m

Note: This formula is only valid if the volume for the reconstitution is 1000 ml (see step 1 in the section Preparation of a-glucosidase). 12350 Calculation factor wmeasurement at 405 nm, ´405(4-NP)s1012 m2Ømol –1x DA Dt

Change in absorbance after correction of the reagent blank rate (s –1).

Fdilution Dilution factor (441 for the example above) m

Weighed mass (mg) of the reconstituted a-glucosidase.

blyo

Catalytic mass concentration wmkatØmg –1x of a-glucosidase in the lyophilized material.

The catalytic mass concentration in mkatØmg –1 can be converted to UØmg –1 by multiplication by the factor fs60. The same procedure is also used to determine the relative inhibition of a-glucosidase caused by the matrix of some a-amylase samples. Two measure-


Table 9 Comparison of the IFCC methods for measurement temperatures of 308C and 378C. 378C Reference procedure

308C Reference method

Comment

Specimen investigated Calibration materials, control specimens and human sera

Human serum

The reference procedure is used primarily for the measurement of control samples and calibration materials

pH of the final complete reaction mixture The optimum pH is 7.00 The optimum pH is 7.10 pH of solution 2 There is no difference from the pH of the reaction mixture

The difference from the pH of the reaction mixture is 0.02 units

Tolerance for the pH adjustment pH "0.03

Not specified

Tolerance for the temperature adjustment Uncertainty F0.18C (ks2) Bias less than "0.058C, imprecision less than "0.18C

The optimum pH depends on the measurement temperature Addition of EPS to the final reaction mixtures lowers the pH in the procedure for 308C

High quality spectrophotometer with devices for temperature adjustment and control should provide temperature uncertainty (ks2) of F0.18C

Incubation time 60 s

At least 300 s

There is no need for a long incubation time of 300 s if the buffer solution and the sample are pre-warmed to 378C. Contact between the sample and buffer should not be unnecessarily long

Measurement time 180 s

At least 300 s

Higher signals at 378C allow a shorter measurement time without increasing the imprecision

Catalytic a-glucosidase concentration 8100 UØl –1

4800 UØl –1

Same amount of a-glucosidase for 308C and 378C. The higher temperature induces an increase in enzyme activity

Preparation of the starting reagent solution The starting reagent solution is The starting reagent solution is prebuffered with HEPES. The pH has to be pared with solution 2 (pH 7.12) as the adjusted to 7.00. The pH of solution 2 solvent. The pH of the final reaction mixis also adjusted to 7.00 ture is 7.10 owing to the acidity of EPS Determination of the inhibition of a-glucosidase by the sample matrix Determination of the relative decrease Did not deal with the problem of in the catalytic concentration of inhibition of a-glucosidase by the a-glucosidase caused by the sample sample matrix matrix is described and maximum allowable inhibition is defined Control of the mass fraction of G7-4-NP in EPS Contamination of EPS by G7-4-NP is Did not deal with the problem of limited to 0.1%. A procedure for G7-4-NP contamination of the substrate checking compliance with this specification is included in the reference procedure

Unit of catalytic enzymatic concentration mkatØl –1 and UØl –1 mkatØl –1

Diluent for the reagent enzyme and preparation of the reaction solution Bovine serum albumin and sodium Lyophilized a-glucosidase is used for chloride in water are used for the preparation of the reaction solution. preparation of stock a-glucosidase The mass equivalent to 600 U necessary solution, which is stored at –258C until for 100 ml of reaction solution is use. The reaction solution is prepared weighed on a micro balance by pipetting 0.25 ml of the a-glucosidase solution into 25 ml of solution 2

The acidity of EPS seems to differ from lot to lot. Therefore, the pH of solution 2 and of the starting reagent solution has to be adjusted separately Relative decreases of 0–60% of the catalytic concentrations of a-glucosidase were observed (investigation by Schumann and Klauke of 17 control materials and calibrators, and 11 human sera) G7-4-NP causes a prolonged lag phase that can be longer than the delay time. Determination of the catalytic concentration of a-amylase is influenced in these cases. Therefore, contamination is limited to 0.1% in the EPS specification UØl –1 is the common unit in clinical chemistry, but mkatØl –1 is based on the SI system The procedure for 378C seems to be simpler. The preparation of volumes smaller than 100 ml of reaction solution is possible without loss of precision of the enzyme content. The a-glucosidase solution is stable for a long time (6 months at –258C)



(Table 9 continued) 378C Reference procedure

308C Reference method

Comment

Sample blank rate Not taken into account

Subtraction

Usually, sample blank rates are not subtracted in routine procedures. Therefore, assigned values for calibrators and control materials are only useful for routine methods if they include the sample blank rate

Temperature of the starting reagent solution before use The starting reagent solution should No information about the temperature reach a temperature of 378C before use Data collection Number of readings G6

No information about the number of measurement points

Determination of the slope (time vs. absorbance) Regression analysis using the least No information squares method Reference range Women and men 0.52–1.78 mkatØl –1 (31–107 UØl –1)

Molar absorption coefficient nitrophenoxide in the reaction mixture The molar absorption coefficient was ´405(4-NP)s1056.8 m2Ømol –1 in the determined as reaction solution at 308C. ´405(4-NP)s1012 m2Ømol –1. ´ is independent of the ´405(4-NP)s975 m2Ømol –1 if samples protein content of the sample with total protein concentrations between 65 and 77 gØl –1 are added to the reaction mixture

Calculation of the matrix-dependent inhibition of a-glucosidase The relative (%) inhibition of the catalytic concentration of a-glucosidase caused by the matrix of the sample in the final complete reaction mixture is calculated from the conversion rates obtained with and without the investigated sample.

B

E DA sample Dt Inhs100 1y DA Sol B D G Dt

C

F

DA Change in absorbance in the presence of Sample the sample investigated instead of Solution Dt B (s –1)

At least 6 readings should ensure sufficient precision of the measurement results A well-defined statistical method is necessary to ensure the reproducibility of the slope calculation

Women 0.41–1.08 mkatØl –1 (24.4–64.8 UØl –1) Men 0.42–1.10 mkatØl –1 (25.2–66.0 UØl –1)

ments are performed: one using Solution B and the other substituting Solution B by the a-amylase sample under investigation. In both measurements, the reaction is started with a solution of a-glucosidase diluted with the Diluent for Reagent Enzymes to a catalytic concentration between 8 mkatØl –1 and 33 mkatØl –1 (500 mØl and 2000 UØl –1).

The use of starting reagent solution at ambient temperature decreases the temperature in the cuvette

Reference values for women and men were investigated separately for the measurement temperature of 378C

The difference can be explained partly by the different pH of the reaction mixtures at 308C and 378C. The reason for the influence of protein at 308C could not be explained. The use of ´405(4-NP) s1012 m2Ømol –1 is recommended by IRMM/IFCC

Change in absorbance using Solution B (s –1) DA Sol B Dt Inh Relative (%) inhibition of the catalytic concentration of a-glucosidase caused by the sample matrix in the final complete reaction mixture Note: If the relative inhibition of the catalytic concentration of a-glucosidase by the test sample exceeds 45%, the lag phase may be not complete during the delay time. Such material should not be recommended as a calibrator or control material.

Appendix 2: Control of the mass fraction of G74-NP in EPS Solution 3 of the reference procedure for a-amylase is diluted 1q4 with the Diluent for Reagent Enzymes. This solution is mixed with the starting reagent solution at 1q100. Another starting reagent solution is mixed at the same ratio with the Diluent for Reagent Enzymes. Both mixtures are incubated at ambient temperature and protected from light for 1 h. The absorbance at 405 nm of the starting reagent solution containing a-glucosidase is measured at 378C. Starting reagent solution without a-glucosidase is used as the reference.


The contamination of EPS by G7-4-NP is F0.1% if the measured absorbance is F0.32. Note: The absorbance value of 0.32 is based on EPS with a content of 100%. Correction has to be performed if the lot of EPS used has a content -100%.

Appendix 3: Changes in the IFCC reference procedure for measurements at 378C compared with the reference method for measurements at 308C as described in the original IFCC document The primary reference procedure is derived from the IFCC reference method (8), which provides optimized conditions for the measurement of catalytic activity concentrations of a-amylase. The measurement temperature of 378C instead of 308C requires only minimal changes of certain measurement parameters to retain the optimum measurement conditions. The modifications are listed and commented on in Table 9. Furthermore, if a more accurate specification in comparison to the 308C reference method has become necessary to improve the high level of standardization of the measurements, it is also described here.

4.

5.

6.

7.

References 1. Siekmann L, Bonora R, Burtis CA, Ceriotti F, Clerc-Renaud P, Fe´rard G, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes. Clin Chem Lab Med 2002;40: 631–4. 2. Schumann G, Bonora R, Ceriotti F, Clerc-Renaud P, Fe´rard G, Ferrero CA, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 2. Reference procedure for the measurement of catalytic activity concentration of creatine kinase wATP: creatine N-phosphotransferase (CK), EC 2.7.3.2x. Clin Chem Lab Med 2002;40:635–42. 3. Schumann G, Bonora R, Ceriotti F, Clerc-Renaud P, Fe´rard G, Ferrero CA, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations

8.

9.

10.

of enzymes at 378C. Part 3. Reference procedure for the measurement of catalytic activity concentration of lactate dehydrogenase wL-lactate: NADq oxidoreductase (LDH), EC 1.1.1.27x. Clin Chem Lab Med 2002;40:636–48. Schumann G, Bonora R, Ceriotti F, Fe´rard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 4. Reference procedure for the measurement of catalytic activity concentration of alanine aminotransferase wL-alanine: 2-oxoglutarate aminotransferase (ALT), EC 2.6.1.2x Clin Chem Lab Med 2002;40:718–24. Schumann G, Bonora R, Ceriotti F, Fe´rard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 5. Reference procedure for the measurement of catalytic activity concentration of aspartate-aminotransferase wL-aspartate: 2-oxoglutarate-aminotransferase (AST), EC 2.6.1.1x. Clin Chem Lab Med 2002;40:725–33. Schumann G, Bonora R, Ceriotti F, Fe´rard G, Ferrero CA, Franck PF, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 6. Reference procedure for the measurement of catalytic activity concentration of g-glutamyltransferase w(g-glutamyl)-peptide: amino acid gglutamyltransferase (GGT), EC 2.3.2.2x. Clin Chem Lab Med 2002;40:734–8. Siekmann L, Bonora R, Burtis CA, Ceriotti F, ClercRenaud P, Fe´rard G, et al. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 378C. Part 7. Certification of four reference materials for the determination of enzymatic activity of g-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase according to IFCC procedures at 378C. Clin Chem Lab Med 2002;40:739–45. Lorentz K, International Federation of Clinical Chemistry (IFCC). Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 9. IFCC method for a-amylase (1,4-a-D-glucan 4-glucanohydrolase, EC 3.2.1.1). Clin Chem Lab Med 1998; 36:185–203. Clinical and Laboratory Standards Institute. Preparation and testing of reagent water in the clinical laboratory; Proposed guideline, 4th ed. Linsinger T, Schimmel H, Pauwels J, Schumann G, Siekmann L. IRMM/IFCC information. Catalytic concentration of a-amylase determined by IFCC reference method at 378C, IRMM/IFCC-460. EUR 19927 EN. IRMM/IFCC, 2001.